Project Summary The projects proposed in this application aim to expand our understanding of how the Nef protein of HIV functions in vivo to promote viral persistence. Nef prevents the immune system from eradicating infected cells and increases viral replication through a variety of mechanisms. The functions of Nef have been studied primarily using laboratory strains of the virus or Nef isolated from the blood of chronically infected individuals. The studies we propose aim to investigate the following aspects of Nef function in vivo: 1.) How Nef sequence and function evolves in the first 6 weeks of infection, 2.) Comparison of Nef sequence and function in tissues versus blood, and 3.) Identification of which Nef sequences or functions contribute most to viral replication. Better understanding of how Nef contributes to viral persistence in vivo will assist efforts to develop more effective vaccine strategies and new drug treatments. The research plan utilizes nef sequences isolated directly from the blood or tissues of HIV-positive subjects. Primary isolates of nef will be obtained at several time points from one group of subjects in the acute stage of infection so we can follow how Nef changes over time during this critical period. In another group of subjects nef sequences will be isolated simultaneously from blood and tissues (e.g. - gut, brain, testes, ovaries, spleen) so we can compare how Nef is working in different anatomical locations. Lastly, the ability of Nef from each of these groups to promote viral replication in primary blood cells relative to each other and relative to a standard laboratory strain of HIV will be compared. By comparing the relative ability of each isolate to promote viral replication we will be able to identify which sequences or functions of Nef are most critical for its role in contributing to persistent infection. Understanding more about how Nef function changes over time and in different anatomical sites in vivo, as well as how different Nef functions contribute to viral replication will help with the development of strategies to combat the persistent, inevitable march of HIV-related disease.